WMCTC Chromatography Workshop: 7 th Feb 2017

1. WMCTC Chromatography Workshop: 7th Feb 2017 Ian J. Shannon School of Chemistry

2. Objectives • General Concepts/ & Terminology • Practical aspects of TLC • dips, artefacts, additives • columns • Advanced techniques • GC • HPLC

3. Concepts & Terminology • Uses • Analysis and Purification • Separation of Components • Stationary Phase • Usually Silica/Alumina • Mobile Phase • Solvent, referred to as “Eluent”

4. TLC – Silica • Silica supported on plate (Aluminium Foil) Solvent Molecule (Mobile Phase) Hydroxyl-terminated Silica (Stationary Phase) Al Foil (Backing Plate)

5. TLC – Paper Chromatography • Water molecules on Surface of Paper/Cellulose Water molecules hydrogen-bond to surface to form Stationary Phase) Solvent (Mobile Phase) moves across surface through capillary action Paper/Cellulose (“Backing Plate”)

6. TLC Uses • Used for analysing mixtures • Monitoring reactions • Analysing column chromatography fractions • e.g. separating menthol and limonene

7. Preparation of a TLC Plate

8. Analysing a Reaction • A + B -> Products

9. Running a TLC Plate

10. What is Happening?

11. Completion of TLC Don’t forget to mark the solvent front with a pencil at the end!

12. Visualisation • UV – commonly 254 nm • Dips – not all spots are visible under UV • Iodine • KMnO4 solution • Vanillin solution • Ninhydrin • FeCl3 solution • Ceric Ammonium Molybdate (CAM) • Sulfuric Acid

13. Reporting TLC Data • Rf –distance travelled by compound divided by distance travelled by eluent • Reported to 2 d.p. • Include eluent & visualisation

14. Solvent Polarity • Common Eluent Systems • Hexane & Diethyl Ether – Low polarity cpds • Hexane & Ethyl Acetate – Mid polarity cpds • Toluene & Acetone – Aromatic cpds • DCM & Methanol – High polarity cpds

15. Effect of Solvent on Rf • In Hexane & Diethyl Ether

16. TLC Video • http://www.rsc.org/learn-chemistry/resource/res00001074/thin-layer-chromatography

17. TLC Troubleshooting A B C E D F

18. TLC Troubleshooting

19. Additives • Carboxylic acids/amines interact strongly with silica • Use additive in the eluent to prevent streaking • Up to 2% v/v (20 mL additive in 1 L eluent) • Acetic acid for carboxylic acids • Triethylamine for amines

20. A-Level Exemplar: Anadin Extra • Anadin Extra • Caffeine, Aspirin, Ibuprofen, Paracetemol • Ethanol or Methanol as Solvent • Ethyl Acetate for TLC • Silica TLC plates & UV Visualisation • Variations/extensions could include different pain relief tablets, and identifying them based on the components present.

21. Gas Chromatography (GC) • Mobile phase is a gas • Analysis only • Very good separation • Must be able to get components into gas phase • No good for bio-molecules

22. GC Video • http://my.rsc.org/video/55

23. Schematic of Gas Chromatograph

24. Choice of Mobile Phase • In GC, the choice of mobile phase is often limited • Often called the Carrier Gas • Inert to samples and instrumentation • Chosen for ease of purification/drying • Examples include N2, Ar, H2 • Accurate flow control/measurement needed to obtain reproducibility

25. Types of Stationary Support • Packed columns have particles with surface coated or bonded stationary phase • Resistance flow restricts the length and hence resolution achievable • Capillary columns have surface coated or bonded stationary phase • As there is little flow resistance (hollow tube!) columns of >20 m are perfectly possible and therefore excellent resolution can be achieved • Not all stationary phases can be used in capillary columns

26. Choice of Stationary Phase • Requirements – Thermally stable, Inert, Low volatility • Polar stationary phase (capillary or packed column) • Non-polar Stationary Phase (capillary or packed column) Intermediate Polarity Stationary Phases are available

27. Chromatogram • A chromatogram is a graph showing the detector response as a function of elution time • The retention time, tr, is the time from the injection of the mixture onto the column until that component reaches the detector. • Eluent travels through the column in the minimum time possible - Designated tm • The adjusted retention time, tr’, for a solute is the additional time for a solute to travel the length of the column tr’ = tr - tm

29. HPLC • High Pressure Liquid Chromatography • High Performance Liquid Chromatography • Allows Analysis and/or Purification

30. HPLC • Small particle size – 2-10 μm (60 μm for ‘normal’ columns) • Greater surface area • High pressure • Needed to achieve sensible flow rate • Forces interactions with stationary phase

31. Normal and Reverse Phase • Normal Phase • Polar compounds move slower • Polar solvents have greater eluting power • Example Solvent: Hexane/iso-propanol • Reverse Phase • Silica is derivatised with a long chain hydrocarbon (C18) • Non-polar compounds move slower • Non-polar solvents have greater eluting power • Example Solvent: Water/acetonitrile

32. Gradient Solvent System • A gradual increase in the proportion of one solvent to increase/decrease the polarity • e.g. 10 % MeCN in water to 40 % MeCN in water over 15 min • Useful for multicomponent systems • Slower sample throughput • Can take longer to optimise

33. Isocratic Solvent System • A single solvent or constant solvent mixture is used for the chromatography • Improves sample throughput as the solvent in the system at the end is the same as at the beginning • Quicker to optimise • No issues relating to solvent mixing